In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industry can accomplish these goals, one of which is chromatography. Chromatography is quite well suited to a variety of uses in the field of biotechnology, since it can separate complex mixtures with great precision and also is suitable for more delicate products, such as proteins, since the conditions under which it is performed are not typically severe.
One chromatography method, which is an especially sensitive separation technique and also applicable to most types of proteins, is metal chelate affinity chromatography (MCAC), also known as immobilised metal ion adsorption chromatography (IMAC). This technique is commonly used in purification schemes together with another chromatographic step, such ion exchange chromatography (IEX) and/or hydrophobic interaction chromatography (HIC).
More specifically, IMAC utilises matrices that comprises a group capable of forming a chelate with a transition metal ion, which chelate in turn is used as the ligand in chromatography to adsorb a compound from a liquid. The binding strength in IMAC is affected predominately by the species of metal ion, the pH of the buffers and the nature of the ligand used. Since the metal ions are strongly bound to the matrix, the adsorbed protein can be eluted either by lowering the pH or by competitive elution.
In general, IMAC is useful for separation of proteins or other molecules that present an affinity for the transition metal ion of the matrix. For example, proteins will bind to the matrix upon the presence of accessible histidine, cysteine and tryptophan residues, which all exhibit affinity for the chelated metal.
With the advent of molecular biological techniques, proteins are now easily tailored or tagged with one or more histidine residues in order to increase their affinity to metal chelated ligands, and accordingly, metal chelate chromatography has more recently assumed a more important role in the purification of proteins.
Simple chelators have been suggested as ligands for IMAC, such as iminodiacetic acid (IDA). IDA, coupled to agarose supports and subsequent charged with various metals, such as Cu2+, Zn2+ and Ni2+, has been used for capture of proteins and peptides and is also available as commercial resins. More specifically, U.S. Pat. No. 4,551,271 (Hochuli, Hoffmann-La Roche Inc.) discloses a metal chelate resin which comprises IDA ligands, in the purification of interferon. The best results are obtained with this resin if the interferon has already been partially purified. The resin can according to the specification be prepared in a known manner by treating agarose with epichlorohydrin or epibromohydrin, reacting the resulting epoxide with iminoacetic acid disodium salt and converting the product into the copper or zinc salt by washing with a copper (II) or zinc solution.
More recently, EP 87109892.7 (F. Hoffmann-La Roche AG) and its equivalent U.S. Pat. No. 4,877,830 (Döbeli et al, assigned to Hoffmann-La Roche Inc.) disclosed a tetradentate chelator known as nitrilotriacetic acid (NTA) for use with metals that have six coordination sites. More specifically, the matrices can be described by the general formula [Carrier matrix]-Spacer-NH—(CH2)x—CH(COOH)—N(CH2COO−)2Ni2+, wherein x=2–4. The disclosed matrix is prepared by reacting an amino acid compound of the formula R—HN—(CH2)x—CH(NH2)—COOH, wherein R is an amino protecting group and x is 2, 3 or 4, with bromoacetic acid in alkaline medium and subsequently, after an intermediate purification step, cleavage of the protecting group and reacting this group with an activated matrix, whereby an amide bond is formed. However, this procedure may involve disadvantages, since the media obtained presents the immobilised desired chelating ligand as well as some unreacted carboxylic groups, thus yielding a heterogeneous media. In addition, the efficiency of the suggested alkylation chemistry is not satisfactory, and after the deprotection step of the amine, the product is not well defined regarding rest products from neutralisation and cleavage.
Finally, WO 01/81365 (Sigma-Aldrich Co.) discloses a metal chelating composition that according to the specification is capable of forming relatively stable chelates with metal ions and exhibits an improved selectivity for polyhistidine tagged proteins. According to said WO 01/81365, the linkage between the chelator and the resin is an important parameter for the selectivity, and the linkage is a neutral ether, a thioether, a selenoether or an amide. The disclosed compositions are coupled to an insoluble carrier, such as Sepharose™ according to given examples. The chromatographic media is produced in two different ways; either by a solid phase reaction directly on to the pre-activated solid support eventually used in the chromatographic media, or by a separate in solution synthesis of the intermediate product N,N,N′,N′-tetrakis(carboxymethyl)-L-cystine that is eventually coupled to the solid support.
Accordingly, there is still a need in this field of alternative methods for synthesis of IMAC ligands and immobilisation thereof to a base matrix.